Figure 1.
The effect of age on post-PNX lung regeneration vs control.
Time course of lung volumes before (day 0 group) and after (days 7, 14, 21) PNX (day 0) in 3, 9 and 24 month mice including A. vital capacity (VC), B. inspiratory capacity (IC), C. dynamic compliance (Cchord) and D. total surface area, computed using TLC and MLI (n = 5 mice/group/time point). * P<0.05 when compared to the day 0 (no PNX) group within age category; † P<0.05 vs. 3-mo control group day 0. Data are expressed as means ± SEM.
Figure 2.
The effect of age on post-PNX lung regeneration vs SHAM.
Comparison of lung volumes in SHAM and PNX mice 21 days after surgery in 3 month and 9 month old animals including A. vital capacity (VC), B. inspiratory capacity (IC), C. dynamic compliance (Cchord) and D. total surface area, computed using water volume displacement and MLI (agar-inflated) (n = 5 mice/group). Fig. 2A–B: * P<0.05 when compared to the SHAM group within age category. Fig. 2D: * P<0.05 vs. 3 mo PNX (no significant difference between the SHAM lung surface area in 9 mo vs 3 mo mice). Data are expressed as means ± SEM.
Figure 3.
The effect of age on post-PNX cell abundance and proliferation.
A. The effect of PNX on abundance of alveolar type-2 epithelial cells expressed as a percentage of nucleated cells which stained for proSP-C in each age-group, including pre (day 0) and post (day 7) PNX (n = 5 mice/group). * P<0.05 when compared to the 3 mo (day 7) group. † P<0.05 when compared to the 3 or 9 month control (day 0) group. ‡P<0.05 when compared to the 24 mo control group. Data are expressed as means ± SEM. B. Percentage of alveolar type-2 cells (AECII) that were Ki67pos in each age-group pre (day 0) and post (day 7) PNX (n = 5 mice/group). * = P<0.05 when compared to the control within age group; † P<0.05 vs. 9 mo post-PNX data. Data are expressed as means ± SEM; C. Percentage of cells in the alveolar region (excluding AECII) that were Ki67pos in each age-group control (day 0) and post (day 7) PNX (n = 5 mice/group). * = P<0.05 when compared to the control (day 0) values. † = P<0.05 when compared to the day 7 post-PNX data for the 3 mo group. There was no significant difference between the 9 and 24 mo groups. Data are expressed as means ± SEM; D. Representative examples of Ki-67 and pro-SP-C staining in the lung parenchyma after (day 7) PNX in 3 and 24 mo mice.
Figure 4.
The effect of age on post-PNX cellular apoptosis.
Apoptosis as a function of age (3, 9, and 24 mo) and PNX (day 0 vs. 7). A. Percentage of nucleated cells that were TUNEL positive; B. Percentage of proSP-C positive (AECII) cells that were TUNEL positive. For A. and B., * P<0.05 compared to day 0 within age group; † P<0.05 compared to d7 in 3 mo old mice; n = 5/group. Data are expressed as means ± SEM; C. Examples of TUNEL and proSP-C immunostaining used for enumeration of single (single line arrows) and double (double line arrows) staining (400×mag).
Figure 5.
Study design comparing transcriptomic patterns after PNX in 9 vs 3 month mice.
A: Surgeries. Left lung lobe pneumonectomy (PNX) was performed on 18 young (12 week old) and 18 middle-aged (9 month old) female mice. The left lobe of every animal was preserved in RNAlater at the time of surgery. The mice were sacrificed and the remaining right lung lobes were removed at 1, 3 or 7 days post-surgery (n = 6/group). RNA was prepared from the R and L lung lobes of every animal. For each animal, RNA from the left lung lobe was used as the normalizing RNA for microarray analysis. B: Microarray experimental design. At each time point, RNA from two animals was pooled to create one pre-PNX (left lung) sample and one post-PNX (right lung) sample. Therefore, at each time point, 3 pooled samples were created for each group (young pre-PNX; young post-PNX; aged pre-PNX and aged post-PNX). C. Microarray data analysis. We generated two groups of data from the microarray analysis. First, to uncover transcripts that are differentially regulated between young and aged animals, we directly compared the aged pre-PNX samples to the young pre-PNX samples (n = 9 pooled samples/group). Second, to compare the effect of aging on lung regeneration post-PNX, we performed a double comparison by first comparing young post-PNX and aged post-PNX to their own control (pre-PNX) for each pooled sample. (n = 6 data sets/time point), and then using a two-class SAM method to compare these aged vs young data sets to identify preferentially expressed genes in aged vs young post-PNX animals.
Table 1.
Validation of the microarray data (day 1 PNX vs SHAM) using quantitative PCR.
Figure 6.
Ingenuity Pathway Analysis (IPA) of transcriptomic patterns without PNX.
Top network illustrates transcripts that are significantly modulated in a whole lung analysis of 9 vs. 3 month mice (no surgery). Green indicates down-regulated transcripts, red indicates up-regulated transcripts. Key: The node shapes denote - enzymes phosphatases
kinases
peptidases
G-protein coupled receptors
transmembrane receptors
cytokines
growth factors
ion channels
transporter
transcription factor
other
.
Figure 7.
Ingenuity Pathway Analysis (IPA) of transcriptomic patterns 1 day after PNX.
Top network illustrate transcripts that are significantly modulated in a whole lung analysis 1 day after PNX in 9 vs. 3 month mice. Green indicates down-regulated transcripts, red indicates up-regulated transcripts. Key: The node shapes denote - enzymes phosphatases
kinases
peptidases
G-protein coupled receptors
transmembrane receptors
cytokines
growth factors
ion channels
transporter
transcription factor
other
.
Figure 8.
Ingenuity Pathway Analysis (IPA) of transcriptomic patterns 3 days after PNX.
Top network illustrates transcripts that are significantly modulated in a whole lung analysis 3 days after PNX in 9 vs. 3 month mice. Green indicates down-regulated transcripts, red indicates up-regulated transcripts. Key: The node shapes denote - enzymes phosphatases
kinases
peptidases
G-protein coupled receptors
transmembrane receptors
cytokines
growth factors
ion channels
transporter
transcription factor
other
.
Figure 9.
Ingenuity Pathway Analysis (IPA) of transcriptomic patterns 7 days after PNX.
Top network illustrates transcripts that are significantly modulated in a whole lung analysis 7 days after PNX in 9 vs. 3 month mice. Green indicates down-regulated transcripts, red indicates up-regulated transcripts. Key: The node shapes denote - enzymes phosphatases
kinases
peptidases
G-protein coupled receptors
transmembrane receptors
cytokines
growth factors
ion channels
transporter
transcription factor
other
.
Figure 10.
Enumeration of aSMA-positive cells after PNX in 9 vs 3 month mice.
A. Number of αSMA-positive cells (per 1000 nucleated cells) 0, 7 and 21 days post-PNX in 3 and 9 month mice. Data are mean values ± SEM. *P<0.05 between Day 7 and Day 0; B. Micrograph illustrating the difference in αSMA-positive cell abundance in the lung parenchyma at day 7 post-PNX in 3 and 9 month mice. Photomicrograph (400× magnification) with αSMA (green) and DAPI nuclei (blue).
Figure 11.
Collagen content after PNX in 3, 9 and 24 month mice.
Picrosirius staining as a measure of collagen content in alveolar parenchyma before and after PNX in 3, 9 and 24 month old mice; *P<0.05 3 month pre vs. post, † P<0.05 9 mos vs. 3 mo pre, ‡ P<0.05 24 mo pre or post vs. 3 or 9 mo pre or post; n = 8 mice/group. Data are expressed as mean ± SEM.
Figure 12.
Comparison of the prevalence of colony forming fibroblasts (CFU-F) in the lung of 3, 9 and 17 month old mice.
A. Examples of representative CFU-F plates derived from 3, 9 and 17 month old mice at passage 5. B. Comparison of passages 0 (primary digest), 3, 5, and 7 CFU-F derived from 3, 9 and 17 month old mice. *P<0.05 compared to same passage fibroblasts from 3 month old donor mice; † P<0.05 17 months vs. 9 months same passage fibroblasts; n = 3 experiments/time point. C. Comparison of mean florescence intensity obtained by flow cytometry as a measure of cell size between 3, 9 and 17 month cultured lung fibroblasts (passage 4). Cells were gated on forward and side-scatter and 7AAD (negative), and then analyzed for forward scatter mean (30,000 independent events). *P<0.05 compared to fibroblasts from 3 month old mice.
Figure 13.
Morphology and phenotype of aging lung fibroblasts.
Comparison of explant-grown lung fibroblasts derived from 3, 9 and 17 month old mice, as well as passage 29 (XP27) cells derived from 3 mo mice, grown in 0% serum (200× phase contrast micrographs), as well as lung fibroblasts positive for αSMA, Col3A1 and S100A4 (red), compared to all nucleated cells (blue) (200× micrographs). Representative isotype controls (using fibroblasts from 17 month mice) are included for reference.
Table 2.
Relative expression (fold change) of select genes in 9 month, 17 month, and late passage (p27) 3 month lung fibroblasts compared to 3 month control lung fibroblasts.
Table 3.
Relative expression (fold change) of select genes in 3, 9, 17 month and passage 27 (derived from 3 month mice) lung fibroblasts after activation by Tgfβ1 compared to un-activated age-matched controls.
Table 4.
Relative expression (fold change) of select genes in 3, 9 17 month and passage 27 (derived from 3 month mice) lung fibroblasts after activation by Tgfβ3 compared to un-activated age-matched controls.